Conventional processes for protein purification typically involve cell culture methods, e.g., using either mammalian or bacterial cell lines recombinantly engineered to produce the protein of interest (e.g., a monoclonal antibody) followed by a cell harvest step to remove cell and cell debris from a cell culture broth. The cell harvest step is usually followed by a capture step, which is typically followed by one or more chromatographic steps, also referred to as polishing steps, which usually include one or more of cation exchange chromatography and/or anion exchange chromatography and/or hydrophobic interaction chromatography and/or mixed mode chromatography and/or hydroxyapatite chromatography. A virus inactivation step may also be included after the capture step. The polishing steps are usually followed by virus filtration and ultrafiltration/diafiltration, which completes the purification process. See, e.g., Shukla et al., J. Chromatography B., 848 (2007) 28-39; Liu et al., MAbs, 2010: September-October 2(5): 480-499.
The virus filtration step is an important step in the purification of proteins, especially when the product is desired to be used in diagnostic or therapeutic applications. The virus filtration step typically employs virus retention filters that retain virus particles while allowing the protein of interest to pass through. One of the challenges with achieving efficient virus filtration is that some of the impurities, especially protein aggregates, have a similar size to the virus particles. Accordingly, protein aggregates end up fouling or plugging the virus retention filter, thereby significantly reducing the flow of the protein of interest through the filter.
Impurities such as protein aggregates form at different stages of the purification process, and in order to maintain adequate throughput of virus retention filters, protein aggregates need to be removed prior to reaching the virus retention filters. Attempts have been made in the prior art to implement flow-through aggregate removal based on Hydrophobic Interactions Chromatography (HIC) media (see, e.g. U.S. Pat. No. 7,427,659). However, HIC-based preparative separations have narrow applicability due to generally difficult process development, narrow operating window, and high concentration of salt required in the buffer.
Weak partitioning chromatography (WPC) is another mode of chromatographic operation, in which the product binds weaker than in the case of bind-elute chromatography but stronger than in the case of flow-through chromatography (See, e.g. U.S. Pat. No. 8,067,182); however, WPC also has certain draw back associated with it including, a narrow operating window and lower binding capacity for impurity removal compared to bind and elute methods.
Further, one of the most convenient ways to selectively remove protein aggregates is by passing a solution of proteins and/or other biomolecules through a filtration media prior to reaching the virus retention filter, whereby the protein aggregates are removed, permitting the protein being purified to flow through onto the viral retention filter. Examples of such filters can be found in U.S. Pat. No. 7,118,675, which discusses filtering a solution of proteins and viruses through an adsorptive depth filter or a charged or surface-modified membrane, prior to the virus filtration step.
Additionally, PCT Publication No. WO2010/098867 discusses using a negatively charged porous medium for removing protein aggregates and PCT Publication No. WO2012/175156 discusses a polyamide-based filter for removing protein aggregates. Lastly, U.S. application Ser. No. 13/783,941, US 20130245139 A1 filed Mar. 4, 2013, discusses use of compositions comprising cation exchange groups for removal of protein aggregates, however, the compositions described therein operate well only under pH and conductivity conditions suitable for cation exchange chromatography.